Plasminogen activators are serine proteases that convert the catalytically inactive zymogen plasminogen to its active form, plasmin, which is required for the dissolution of fibrin. Because of this property, several plasminogen activators are now being used as in-vivo fibrinolytic agents in the treatment of acute myocardial infarction. One such plasminogen activator is tissue plasminogen activator, which has been the focus of considerable attention because of its enhanced fibrinolytic activity in the treatment of acute myocardial infarction and pulmonary embolism.
Human tissue plasminogen activator, a serine protease containing 527 amino acids with 17-pairs of S—S cross-links is a single polypeptide chain glycoprotein with a molecular mass of about 70 kDa. The glycosylated tPA is found to appear with two different forms—Type I and Type II. The carbohydrate moiety mainly comprises of N-linked and O-linked glycosylations at the positions Asn448, Asn117 and Asn184 (N-linked) and Thr61 (O-linked) glycosylation and contributes about 7% of the total molecular weight of the protein. Type I tPA is known to contain glycosylated moiety on all the above specified residues, whereas Type II population of tPA does not exhibit glycosylation at the position Asn184 (Kazuya Mori Jr Biol. Chem. 1995 Feb. 17; 270(7):3261-7). The isolated human tPA exhibits an isoelectric pH of about 7.5. Although tPA is synthesized inside the cell as a single-chain polypeptide, it has been reported that upon releasing in to the extracellular medium tPA gets proteolytically cleaved by plasmin at the C-terminus of Arg275 (Kazuya Mori J Biol. Chem. 1995 Feb. 17; 270(7):3261-7). Proteolytic cleavage converts the single chain tPA in to a two-chain form which remains held together by a single S—S bond. However, it is known in the art that the single- and double-chain forms of tPA do not show any significant difference in fibrinolytic activity.
TNKase (TNK-tPA), a recombinant form human tPA is produced in CHO cells as a secretary protein and is known to play the same role as human tPA. Recombinant tPA is used as a therapeutic agent for the treatment of acute myocardial infarction. TNK-tPA has three sets of mutations on its polypeptide backbone at the positions T103N,N117Q, KHRR(296-299)AAAA, as demonstrated in (Proc. Natl. Acad. Sci. Vol. 91, pp. 3670-3674, April 1994) and also in U.S. Pat. No. 5,612,029. Similar to that of the human tPA, the recombinant TNK-tPA also consists of Type I and Type II glycoforms and has been observed to appear as a mixture of single- and double-chain form.
It has been reported that single chain species of recombinant tPA (produced by CHO) with more than 70% purity of the single-chain form remains stable for more than 2.5 y when stored in lyophilized form, under room temperature conditions (Journal of Interventional cardiology, Vol 2, No 2, 1989). It has been also demonstrated that the double chain species of recombinant tPA (cleaved form) obtained from CHO cells remains fairly stable in liquid formulation for at least 1 year, when stored between 0-4° C. These observations indicate that both the single-chain and double-chain forms of tPA remain reasonably stable under various storage conditions.
For commercial purpose, recombinant tissue plasminogen activator or TNK-TPA has been overexpressed in CHO cells and purified with the help of various column chromatography steps. These chromatography steps mostly comprise of certain affinity column chromatography matrices to isolate tPA with its species variants like Type I and Type II in a selective manner. One such widely used affinity chromatography is Lysine-sepharose column chromatography. Lysine-sepharose matrix binds to tPA by the lysine-binding site of its kringle 2 domain. Therefore, this column step has been found to be useful in capturing the tPA molecule in a selective manner from crude mixture during column purification (Byeon I J, Kelley R F, Llinas M. Eur J. Biochem. 1991 Apr. 10; 197(1):1155-65). Further, U.S. Pat. No. 4,898,825 disclose the use of Erythrina trypsin inhibitor as an affinity agent for the purification of tPA. U.S. Pat. No. 5,411,864 discloses the use of antibodies for the purification of tPA. Similarly, U.S. Pat. No. 5,015,583 discloses the use of heparin sepharose affinity column for the purification of tPA. U.S. Pat. No. 5,141,863 disclose the use of hydroxyapatite column for the purification of tPA.
Hence, a wide variety of chromatography processes, e.g. affinity and hydroxyapatite, have been applied for the purification of tPA. However, the cost of affinity or hydroxyapatite matrices is very high and therefore, not very cost effective for industrial use. Besides, none of the process has been reported yet to give rise to a desired mixture of the single- and double-chain forms of tPA while removing the excess amount of double chain form of the said protein during purification. In the absence a suitable process, the process of tPA purification remains a challenge. Hence, there is a need to develop an industrially viable, cost effective process for the purification of the desired tPA preparation.
Thus, the present invention solves the problem associated with the process described in the prior art and also brings scientific advancement which makes the present process advantageous over the processes known in the prior art related to the purification of tPA in its desired form. In the present purification process, tPA is captured first from a crude mixture without any significant loss of the desired protein and is eluted in a differential manner from a hydrophobic column in the presence of a suitable organic solvent while removing the undesired contaminating proteins and species variants.